The mechanisms of cancer development in children are poorly understood. Molecular analysis of the recurrent chromosomal translocations found in many pediatric solid tumors has identified unique fusion oncogenes for each tumor type. Ewing's sarcoma is a pediatric tumor of uncertain histologic origin that is defined by the presence of a specific chromosomal rearrangement, t(11;22)(q24;q12). This translocation generates the EWS/FLI fusion oncogene. While EWS/FLI expression appears to be required for Ewing's sarcoma development, it is unlikely to be the only genetic alteration present in this tumor. The goal of this proposal, therefore, is to identify additional genetic events that are required in the genesis of Ewing's sarcoma. While expression of EWS/FLI in immortalized murine fibroblasts results in cellular transformation, our preliminary work demonstrates that expression of EWS/FLI in primary human fibroblasts results in growth arrest. Our hypothesis is that primary cells have growth-inhibitory pathways in place that prevent neoplastic transformation by oncogenes, and that these pathways are likely to be abrogated in Ewing's sarcoma. We propose a multifaceted approach to identify these pathways. First, we will use data obtained from expression analysis to identify and validate pathways that mediate this growth arrest. Next, we will identify new pathways by suppressor screening. Finally, we will determine whether these pathways are altered in Ewing's sarcoma clinical samples to identify which are biologically relevant to the development of this tumor. The results generated by these studies will not only identify cooperative mutations important for Ewing's sarcoma development, but will also provide unique insights into the mechanisms that primary cells use to inhibit oncogenic transformation.